Our objective is to understand the factors regulating de novo DNA methylation in normal cells and during development and determine how these control pathways go awry in tumor cells and contribute to tumorigenesis. DNA methylation, or the covalent addition of a methyl group to the 5-position of cytosine in the context of the CpG dinucleotide, is required for mammalian development, has profound effects on chromatin structure, and has roles in both DNA repair and genome stability. It has become clear that while essential for development, DNA methylation patterns become dysregulated in cancer, with a generalized genome-wide hypomethylation (loss) in combination with region-specific hypermethylation (gain), primarily at CpG islands. In addition, DNA hypermethylation has been shown to efficiently and heritably inactivate genes. When this occurs in the promoter region of a growth-regulatory gene it can give that cell a growth advantage and ultimately lead to cancer. Mechanisms for the establishment or targeting of methylation patterns in normal cells are almost completely unknown and thus we have focused our studies on the regulation of the cellular enzymatic methylation machinery, the DNA methyltransferases (DNMTs).. Methylation clearly can be targeted since certain genomic regions are always heavily methylated, like pericentromeric heterochromatin (regions of DNA adjacent to the centromere) while regulatory regions for many essential genes are always unmethylated. We have taken a biochemical approach to identify factors that interact with the DNMTs that may alter their nuclear localization and enhance or inhibit their enzymatic activites on particular DNA substrates. Our studies reveal that the most abundant methyltransferase, DNMT1, interacts with the retinoblastoma gene product (Rb). Rb is a critical regulator of cell division and helps a cell decide whether to divide or to go into a semi-permanent resting state. Rb, or other components of the Rb pathway, are mutated in almost all tumor cells. We have determined that cancer-associated mutations in Rb abolish the ability of Rb to interact with DNMT1. Thus we may have identified one mechanism whereby methylation patterns become disrupted in tumors due to an inability of Rb to interact with DNMT1. The identification of factors that interact with and direct methylation in normal cells opens up exciting new possibilities for understanding tumor development and may ultimately lead to novel therapies designed to restore normal methylation patterns and growth control.